Measure intensities in single-cells in two channels with spectral unmixing and using post-hoc seed image for segmentation

Parameters:

Name	#	Type	Description
Channel Number Probe #1 (cell shape is determined from this; (Multi-Dimensional Open #1)	1	integer	Channel number associated with the image, as appears in the Multi-Dimensional Open Dialog #1. Use Edit/Rename to view or change channel number.
Channel Number Probe #2 (Multi-Dimensional Open #1)	2	integer	Channel number associated with the image, as appears in the Multi-Dimensional Open Dialog #1. Use Edit/Rename to view or change channel number.
Channel Number for post-hoc nuclear marker (Multi-Dimensional Open #2)	3	integer	Channel number associated with the image, as appears in the Multi-Dimensional Open Dialog #2. Use Edit/Rename to view or change channel number.
Local background subtraction by median rolling ball	4	boolean	Rolling ball-style background subtraction will be performed: the median of the image is subtracted from the image
Rolling ball: Median filter size (pixels)	5	integer	The median is calculated over a width x width rectangular area around each pixel of the image.
Background Level (Percentile)	6	real	Background is calculated as frame-by-frame median of those pixels that are the darker than this percentile of image histogram for the duration of the entire recording. This has no effect if rolling ball is used.
Spectral Unmix Coefficient Matrix	7	string	To determine spectral crossbleed coefficients use the Tools/Calculate Crossbleed Correction Factor main menu point, or the Math/Blind Spectral Unmix with NMF function. See more on the layout of the Spectral Unmix Coefficient Matrix in the description of Math/Spectral Unmix.
Channel alignment based on only the first frame	8	boolean	Calculates shift from first few frames, then aligns the whole time lapse. No aligns each frame of the time lapse. The number of frames is set by the value used for ΔF/F0 averaging in the Preferences
Image stabilizer compares to first frame	9	boolean	This is the fastest way of image stabilization. Turn it off if the image changes a lot during the time lapse, and it is not possible to compare to the first frame any more.
Image stabilizer period (frames)	10	integer	1 if comparing to first frame. Use more frames if not comparing to first frame and the view filed moves slowly.
ROI drawing: debris cutoff for seed detection (percentile)	11	real	This percentile of the image histogram sets the intensity value where the maximum of the Look Up Table (LUT) is scaled. Use -1 to override this with fixed value set below at "Max value".
ROI drawing: debris cutoff for cell shape detection (percentile)	12	real	This percentile of the image histogram sets the intensity value where the maximum of the Look Up Table (LUT) is scaled. Use -1 to override this with fixed value set below at "Max value".
ROI drawing: approximate diameter of nuclei	13	integer	Diameter of the nucleus in pixels
ROI drawing: minimum nucleus fluorescence (%)	14	real	Cells dimmer than this in filtered rescaled images will be rejected. Increase this value if debris dimmer than the cells is detected.
ROI drawing: cell size, minimum area (pixels)	15	integer	Volume in 3D, area in 2D. 0 for not checking
ROI drawing: cell size, maximum area (pixels)	16	integer	Volume in 3D, area in 2D. 0 for not checking
ROI drawing: minimum cell fluorescence (%)	17	real	Cells dimmer than this in filtered rescaled images will be rejected. Increase this value if debris dimmer than the cells is detected.
ROI drawing: weld segments into round objects	18	boolean	Weld touching segments if they form a rounder object together. Use this to avoid objects fragmenting into multiple segments.
Save single cell fluorescence traces	19	boolean	Saves output as TAB delimited text file
Save file name for probe #1 (for txt file output)	20	string	See main menu Help/"Help on Expression Evaluation" for automatic file naming.
Save file name for probe #2 (for txt file output)	21	string	See main menu Help/"Help on Expression Evaluation" for automatic file naming.
Plot normalization	22	string	This option supersedes ΔF/F0 normalization.
Show results in microplate format	23	boolean	Organizes data in table according to microplate well associations in additional worksheets.

Description:

This pipeline analyzes a two-channel fluorescence time-lapse recording on the single-cell level, using a post-hoc nuclear stain. For the post-hoc recording the same view field is captured with the addition of a nuclear stain e.g. Hoechst 33342 after the functional time lapse has been concluded (this is an additional channel to the two probe-channels during live imaging). The pipeline loads the functional time lapse from Multi-Dimensional Open #1 (tag the dialog in the top right corner) and the post-hoc stain image form Multi-Dimensional Open #2. The pipeline creates a single segmented image depicting the location of each identified cell based on the nuclei and the fluorescence of probe #1. Fluorescence intensities are measured in stabilized, background subtracted and spectrally unmixed images of the original probe fluorescence. The output is saved as text files (or alternatively can be entered into the Excel Data Window.
To determine spectral crossbleed coefficients use the Tools/Calculate Crossbleed Correction Factor main menu point, or the Math/Blind Spectral Unmix with NMF function. See more on the layout of the Spectral Unmix Coefficient Matrix in the description of Math/Spectral Unmix.

Internal version: V11
V2
Minimum cell fluorescence classifier is also applied for the seeds, speeding up segmentation.
Segment welding is used preventing oversegmentation.
Local background subtraction option.
V3
Changed plotting property: Place channels into columns